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A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata
D M Wolk1, C H Johnson, E W Rice
1University of Arizona, Tucson, Arizona 85721, USA.
Abstract:
The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID(50)) and a minimal infective dose (MID) for E. intestinalis. The TCID(50) is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID(50) have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25 degrees C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log(10) reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.
Insights
Chlorine disinfection effectively inactivates Encephalitozoon intestinalis spores, a waterborne pathogen. A 3-log reduction in infectivity was achieved with 2 mg/L chlorine after 16 minutes, suggesting its utility in water treatment.
Area of Science:
- Environmental Microbiology
- Waterborne Pathogen Research
- Disinfection Byproducts
Background:
- Microsporidia are emerging waterborne pathogens with poorly understood disinfection resistance.
- Encephalitozoon intestinalis, a human gut microsporidian, serves as a model for studying disinfection efficacy.
- Assessing the impact of routine water disinfection methods on microsporidian viability is crucial for public health.
Purpose of the Study:
- To evaluate the effect of chlorine disinfection on the infectivity and viability of Encephalitozoon intestinalis spores.
- To establish quantitative dose-response data for microsporidian spore infectivity using cell culture methods.
- To compare spectrophotometric and hemacytometer methods for spore counting in disinfection studies.
Main Methods:
- In vitro growth of Encephalitozoon intestinalis was used to generate spore inoculums.
- Optimized rabbit kidney cell culture system to determine 50% tissue culture infective dose (TCID50) and minimal infective dose (MID).
- Spores were exposed to varying chlorine concentrations (0-10 mg/L) and contact times (0-80 min) at 25°C and pH 7.
Main Results:
- A 3-log reduction (99.9% inhibition) in Encephalitozoon intestinalis MID was achieved at 2 mg/L chlorine with a 16-minute exposure.
- Spectrophotometric spore counts demonstrated equivalent log(10) reduction results compared to traditional hemacytometer counts.
- Established standardized MID and TCID50 values for Encephalitozoon intestinalis, previously unreported for microsporidia.
Conclusions:
- Chlorine disinfection at 2 mg/L for 16 minutes is effective in reducing Encephalitozoon intestinalis infectivity.
- Spectrophotometric methods offer a viable and less labor-intensive alternative for spore quantification in disinfection studies.
- Findings support the potential of chlorine-based water treatment strategies for controlling microsporidian contamination.
